Airways are defended by a tightly regulated system of mucus clearance whose efficiency is a function of ciliary beat frequency (CBF), mucin secretion rates, and airway surface liquid (ASL) volume, i.e., "hydration". Data from a number of studies, including mouse models and genetic diseases of the human lung, have indicated a surprising dominance of ASL volume as the rate-limiting variable for mucus clearance. Given the importance of ASL volume for to pulmonary health, and the absence of an integrated formulation to describe the mechanisms of ASL volume homeostasis, the overarching hypothesis of the PPG is that ASL volume homeostasis is controlled by density of CFTR and ENaC at the apical membrane and the regulation of their activity provided by the nucleotides and nucleosides contained within ASL. To investigate this hypothesis, we propose four Projects: Project I, "CFTR in Ciliated Airway Cells: Copy Number, Lifetime and Activation State", J. Riordan, P.I., will focus on the regulation of the density (N) of CFTR channels as controlled by ASL volume; 2) Project II, "Regulation of ENaC by Proteases and SGK1", M.J. Stutts, P.I., will focus on the regulation of activity (Po) of ENaC in the apical membrane, as mediated by extracellular proteases and ASL nucleotides; 3) Project III, "Mechanisms of Nucleotide Release by Airway Epithelia", E.R. Lazarowski, P.I., will study mechanisms of constitutive and regulated release of ATP on airway epithelial surfaces, focusing on vesicular trafficking; 4) Project IV, "The Role of Airway Surface Liquid Nucleotides/Nucleosides in Volume Homeostasis", R.C. Boucher, P.I., will integrate the concepts of nucleotide release, CFTR/ENaC density/activity, and ASL volume homeostasis in vitro and in vivo, in experimental and mathematical models of ASL volume homeostasis. The PPG is supported by four Cores: 1) the Administrative Core (A); 2) the Molecular Core (B); 3) the Cell Culture Core (C); 4) an Imaging Core (D). Relevance to Public Health: The long-range goal of the PPG is to provide an integrated, quantitative formulation of how ASL volume is controlled in the mammalian lung in health and in disease. A comprehensive knowledge of these mechanisms should yield important insights into the mechanisms of airways dysfunction in disease, e.g., COPD, CF, and asthma, and lead to novel therapies to hydrate airway surfaces to clear the mucus plugs/plaques that produce airways obstruction and infection in these major pulmonary diseases.